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United States Patent |
6,251,887
|
Heijmans
,   et al.
|
June 26, 2001
|
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives
Abstract
The invention relates to 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane
derivatives having general formula (I), wherein R.sub.1 is (H,OR),
(H,OSO.sub.3 H) or NOR; with R being H, (C.sub.1-6)alkyl or
(C.sub.1-6)acyl; each of R.sub.2 and R.sub.3 is independently hydrogen or
(C.sub.1-6)alyl; n is 0, 1, or 2; X is O, S, S(O) or S(O).sub.2 ; m is 0
or 1; each of R.sub.4, R.sub.5 and R.sub.6 is independently hydrogen,
hydroxy, (C.sub.1-4)alkoxy, halogen, NR.sub.7 R.sub.8 or (C.sub.1-4)alkyl,
optionally substituted by hydroxy, alkoxy, halogen or oxo; each of R.sub.7
and R.sub.8 is independently hydrogen or (C.sub.1-4)alkyl, and wherein the
dotted lines indicate a .DELTA..sup.7 or a .DELTA..sup.8 double bond, or a
pair of conjugated double bonds selected from .DELTA..sup.7,14,
.DELTA..sup.8,14 and .DELTA..sup.6,8(14) ; or a pharmaceutically
acceptable salt thereof. The compounds of the invention have meiosis
regulating activity and can be used for the control of fertility.
##STR1##
Inventors:
|
Heijmans; Christian (Breda, NL);
van der Louw; Jaap (Oss, NL)
|
Assignee:
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Akzo Nobel N.V. (Arnhem, NL)
|
Appl. No.:
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581749 |
Filed:
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June 16, 2000 |
PCT Filed:
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December 11, 1998
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PCT NO:
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PCT/EP98/08130
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371 Date:
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June 16, 2000
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102(e) Date:
|
June 16, 2000
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PCT PUB.NO.:
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WO99/32506 |
PCT PUB. Date:
|
July 1, 1999 |
Foreign Application Priority Data
Current U.S. Class: |
514/183; 514/163; 552/515; 552/516; 552/557; 552/611; 552/633; 552/635 |
Intern'l Class: |
C07J 007/00; C07J 009/00; C07J 051/00; C07J 031/575; C07J 003/00; A61K 031/33; A61K 031/00 |
Field of Search: |
514/183
552/516,557,611,633,635
|
References Cited
Foreign Patent Documents |
WO 96 00235 | Jan., 1996 | WO.
| |
WO 96 27658 | Sep., 1996 | WO.
| |
WO 97 00884 | Jan., 1997 | WO.
| |
WO 98 52965 | Nov., 1998 | WO.
| |
WO 98 55498 | Dec., 1998 | WO.
| |
Other References
Byskov Et Al., "Chemical Structure of Sterols that Activate Oocyte
Meiosis," Nature, vol 374, No. 6522, Apr. 1995, pp. 559-562.
Dygos Et Al., "Steroidal Ethers. Analogues of 7-Ketocholesteral," Journal
of Organic Chemistry, vol. 44, No. 10, May 1979, pp. 1590-1596.
|
Primary Examiner: Qazi; Sabiha N.
Attorney, Agent or Firm: Blackstone; William M.
Parent Case Text
This application is a 371 of PCT/EP98/08130, Dec. 11, 1998.
Claims
What is claimed is:
1. A 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative having
the general formula [I]
##STR9##
wherein R.sub.1 is (H,OR), (H,OSO.sub.3 H) or NOR, with R being H,
(C.sub.1-6)alkyl or (C.sub.1-6)-acyl; each of R.sub.2 and R.sub.3 is
independently hydrogen or (C.sub.1-6) alkyl; n is 0, 1 or 2; X is O, S,
S(O).sub.2 ; m is 0 or 1; each of R.sub.4, R.sub.5 and R.sub.6 is
independently hydrogen, hydroxy, (C.sub.1-4)alkoxy, halogen, NR.sub.7
R.sub.8 or (C.sub.1-4)alkyl and each of R.sub.7 and R.sub.8 is
independently hydrogen or (C.sub.1-4)alkyl; and wherein the dotted lines
indicate a .DELTA..sup.7 or a .DELTA..sup.8 double bond, or a pair of
conjugated double bonds selected from .DELTA..sup.7,14, .DELTA..sup.8,14
and .DELTA..sup.6,8(14) ; or a pharmaceutically acceptable salt thereof.
2. The 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative
according to claim 1, wherein n is 0 or 1, X is O, S, S(O) or S(O).sub.2
and m is 0 or 1.
3. The 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative
according to claim 2, wherein R.sub.1 is (H,OR), and R is H or
(C.sub.1-6)acyl, the dotted lines indicate a .DELTA..sup.8,14 diene
system, and the configuration of the 3-OR substituent is the
.beta.-configuration.
4. A 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative selected
from the group consisting of
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-phenoxypregna-8,14-dien-3-ol,
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-(phenylmethoxy)pregna-8,14-dien-3-o
l,
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(2-methylphenoxy)pregna-8,14-di
en-3-ol and
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylsulfonyl)pregna-8,14-die
n-3-ol.
5. A method for regulating meiosis comprising administering an effective
amount of the 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane
derivative of claim 1 or a pharmaceutically acceptable salt thereof.
6. A pharmaceutical composition comprising the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane of claim 1 or a
pharmaceutically acceptable salt thereof, in admixture with
pharmaceutically acceptable auxiliaries.
7. A method for controlling fertility comprising administering an effective
amount of the 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane
derivative of claim 1 or a pharmaceutically acceptable salt thereof.
8. The 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative
according to claim 1, wherein each of the R.sub.4, R.sub.5 and R.sub.6 is
independently substituted by hydrogen, alkoxy, halogen or oxo.
Description
The invention relates to 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane
derivatives, to pharmaceutical compositions containing the same, as well
as to the use of these 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane
derivatives for the preparation of a medicament for the control of
fertility.
Sexual reproduction involves a cyclic alternation of diploid and haploid
states: diploid cells divide by the process of meiosis to form haploid
cells, and the haploid cells fuse in pairs at fertilization to form new
diploid cells. The process of meiosis is characterized by two meiotic
divisions, unique to both male and female germ cells. During the process
two cell divisions, following one round of DNA replication, give rise to
four haploid cells from one single diploid cell. Chromosomal crossover
events, during which paternal and maternal genetic material is exchanged,
occur during the prophase of the first meiotic division. At the end of the
first meiotic division one member of each chromosome pair, composed of two
sister chromatids is distributed to each daughter cell. The second meiotic
division segregates each sister chromatide into a separate haploid cell.
Male and female germ cells are subject to similar meiotic divisions but
differ in the regulation of these processes.
In the male meiosis is a continuous process in germ cells derived from a
population of immature germ cells, the stem cell spermatogonia. After
sexual maturation of the male, spermatogonia from this stem cell
population embark on meiosis. The first and second meiotic division
proceed without interruption and eventually give rise to four mature
spermatozoa.
In the female, primary oocytes start the first meiotic division already
during the embryonic stage but they remain arrested in the prophase
(dictyate stage) until the female becomes sexually mature. Meiosis resumes
at the time of ovulation (egg maturation) after which the first meiotic
division is completed and the second meiotic division is initiated. In
most vertebrates the second meiotic division is arrested at the metaphase
and only completed after fertilization. At the end of the first and of the
second meiotic division the cytoplasm divides asymmetrically to produce
two secondary oocytes, each with a haploid number of single chromosomes,
but greatly differing in size: one is a small polar body, which eventually
degenerates, and the other is a large cell containing all the
developmental potential. Finally one mature ovum is produced.
The stage at which the developing oocyte is released from the ovary and is
ready for fertilization differs in different species. In both
invertebrates and vertebrates ovarian accessory cells respond to
polypeptides (gonadotropins) produced elsewhere in the body so as to
control the maturation of the oocyte and eventually (in most species)
ovulation. In humans the primary oocytes of the newborn female are
arrested in prophase of meiotic division I and most are surrounded by a
single layer of follicle cells; such an oocyte with its surrounding cells
constitute the primordial follicle. A small portion of primordial
follicles sequentially begin to grow to become developing follicles: the
follicle cells enlarge and proliferate to form a multilayered envelope
around the primary oocyte; the oocyte itself enlarges and develops the
zona pellucida, an extracellular matrix consisting largely of
glycoproteins, and cortical granules, specialized secretory vesicles just
under the plasma membrane in the outer region, the cortex, of the egg
cytoplasm [when the egg is activated by a sperm, these cortical granules
release their contents by exocytosis; the contents of the granules act to
alter the egg coat so as to prevent other sperms from fusing with the
egg].
The developing follicles grow continuously and some of them develop a
fluid-filled cavity, or antrum, to become antral follicles. Development of
such follicles is dependent on gonadotropins (mainly follicle stimulating
hormone -FSH) secreted by the pituitary gland and on estrogens secreted by
the follicle cells themselves. Starting at puberty, a surge of secretion
by the pituitary of another gonadotropin, luteinizing hormone (LH),
activates a single antral follicle to complete its development: the
enclosed primary oocyte matures to complete the meiotic division I as the
stimulated follicle rapidly enlarges and ruptures at the surface of the
ovary, releasing the secondary oocyte within. As is the case with most
mammals, the secondary oocyte is triggered to undergo division II of
meiosis only if it is fertilized by a sperm.
Studies on the mechanisms controlling initiation and regulation of the
meiotic process in male and female germ cells suggest a role for cyclic
nucleotides in mediating meiotic arrest. Spontaneous maturation of oocytes
can be prevented by compounds that maintain elevated cAMP levels [Eppig,
J. and Downs, S. (1984) Biol. Reprod. 30: 1-11]. Purines, like adenosine
or hypoxanthine, are thought to be involved in the cAMP mediated
maintenance of meiotic arrest [Eppig, J., Ward-Bailey, P. and Coleman, D.
(1985) Biol. Reprod. 33: 1041-1049]. The presence of a meiosis regulating
substance in a culture system of fetal mouse gonads was first described by
Byskov, A. et al (1976) Dev. Biol. 52: 193-200. It was suggested that the
concentrations of a meiosis activating substance (MAS) and a meiosis
preventing substance (MPS) regulate the meiotic process in concert
[Byskov, A. et al. (1994). In "The physiology of reproduction", Eds.
Knobil, E. and Neill, J., Raven Press, New York]. More recently
(3.beta.,5.alpha.,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol (FF-MAS),
isolated from human follicular fluid, and
(3.beta.,5.alpha.,20R)-4,4-dimethyl-cholesta-8,24-dien-3-ol, isolated from
bull testes, were identified by Byskov, A. et al [(1995), Nature 374:
559-562] as endogenous meiosis activating substances in human and bovine,
respectively. These sterols proved to be able to activate the resumption
of meiosis in cultured cumulus enclosed and naked mouse oocytes.
Derivatives of the endogenous sterols, having either a saturated or an
unsaturated cholestane side chain, have been disclosed in the
international patent applications WO 96/00235, WO97/00883, WO97/00884 and
WO98/28323 (NOVO NORDISK A/S) as meiosis regulating substances. Meiosis
regulating substances are compounds that are agonists or antagonists of a
naturally occurring meiosis activating substance. Thus, they might be used
in the treatment of infertility or for contraception.
A drawback of the compounds described in the patent applications mentioned
above is that they are prone to rapid deactivation in the body [Hall, P.
F. (1985) Vitamins and Hormones, 42: 315], thereby restricting their
therapeutic potential as fertility control agents.
A need therefore exists for regulators of the meiotic process having
improved in vivo activity.
To this end the invention provides
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives having the
general formula I
##STR2##
wherein
R.sub.1 is (H,OR), (H,OSO.sub.3 H) or NOR; with R being H, (C.sub.1-6)alkyl
or (C.sub.1-6)acyl;
each of R.sub.2 and R.sub.3 is independently hydrogen or (C.sub.1-6)alkyl;
n is 0, 1 or 2;
X is O, S, S(O) or S(O).sub.2 ;
m is 0 or 1;
each of R.sub.4, R.sub.5 and R.sub.6 is independently hydrogen, hydroxy,
(C.sub.1-4)alkoxy, halogen, NR.sub.7 R.sub.8 or (C.sub.1-4)alkyl,
optionally substituted by hydroxy, alkoxy, halogen or oxo; each of R.sub.7
and R.sub.8 is independently hydrogen or (C.sub.1-4)alkyl;
and wherein the dotted lines indicate a .DELTA..sup.7 or a .DELTA..sup.8
double bond, or a pair of conjugated double bonds selected from
.DELTA..sup.7,14, .DELTA..sup.8,14 and .DELTA..sup.6,8(14) ;
or a pharmaceutically acceptable salt thereof.
It has been found that the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives having the
general formula I show improved meiosis regulating activity. The invention
further provides a pharmaceutical composition comprising a
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative having the
general formula I. A further aspect of the invention resides in the use of
a 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative having the
general formula I for the manufacture of a medicament for the control of
fertility.
The term (C.sub.1-6)alkyl as used in the definition of formula I means a
branched or unbranched alkyl group having 1-6 carbon atoms, like hexyl,
pentyl, butyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl and
methyl. Likewise, the term (C.sub.1-4)alkyl means an alkyl group having
1-4 carbon atoms.
The term (C.sub.1-6)acyl means an acyl group derived from a carboxylic acid
having from 1-6 carbon atoms, like hexanoyl, pentanoyl, pivaloyl, butyryl,
propanoyl, acetyl and formyl. Also included within the definition of
(C.sub.1-6)acyl are acyl groups derived from dicarboxylic acids, like
hemi-glutaroyl, hemi-succinoyl, and hemi-maloyl. A preferred
(C.sub.1-6)acyl group is hemi-succinoyl.
The term (C.sub.1-4)alkoxy means an alkyloxy having 1-4 carbon atoms, like
butyloxy, propyloxy, isopropyloxy, ethyloxy, and, preferably, methyloxy.
The term halogen means F, Cl, Br or I. When halogen is a substituent at an
alkyl group, like in the definition R.sub.4, R.sub.5 and R.sub.6, Cl and F
are preferred, F being most preferred.
It is understood that the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention have the natural configurations 5.alpha., 9.alpha., 10.alpha.,
13.alpha., 14.alpha.. Preferred agonistic compounds according to the
invention are the 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane
derivatives of formula I wherein n is 0 or 1, X is O and m is 0 or 1. More
preferred are the compounds wherein in addition R.sub.1 is (H,OR), wherein
R has the previously given meaning, and the dotted lines indicate a
.DELTA..sup.8,14 diene system. Among these preferred compounds those with
the 3-OR substituent in the .beta.-configuration are especially preferred.
The configuration at position 20 of the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention can be either R or S. A specifically preferred agonistic
compound of the invention is the 17.beta.-aryloxyalkyl-androstane
derivative
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-phenoxypregna-8,14-dien-3-ol.
Preferred antagonistic compounds according to the invention are the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of formula
I wherein n is 0 or 1, X is S, S(O) or S(O).sub.2 and m is 0 or 1. More
preferred are the compounds wherein in addition R.sub.1 is (H,OR), wherein
R has the previously given meaning, and the dotted lines indicate a
.DELTA..sup.8,14 diene system. Among these preferred compounds those with
the 3-OR substituent in the .beta.-configuration are especially preferred.
The configuration at position 20 of the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention can be either R or S. A specifically preferred antagonistic
compound of the invention is the 17.beta.-arylsulfonylalkyl-androstane
derivative
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylsulfonyl)pregna-8,
14-dien-3-ol.
The meiosis activating activity of the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention is measured in an in vitro oocyte assay as the ability to
overcome the hypoxanthine maintained meiotic arrest in denuded oocytes
(DO) or cumulus enclosed oocytes (CEO).
The meiosis inhibiting activity of the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention is measured in an in vitro oocyte assay as the ability to
inhibit the FF-Mas or 22S-hydroxy-FF-Mas induced maturation, in
hypoxanthine media, in denuded oocytes or cumulus enclosed oocytes,
respectively. The compounds can be used to stimulate or inhibit meiosis in
both male and female and thus can be used as fertility regulating agents.
Fertility regulation comprises contraception and infertility treatment.
For female contraception a
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative according to
formula I can be used for induction of premature maturation of oocytes
which are still inside the ovary, before the naturally occurring
gonadotropin surge [reduced fertility by inducing premature maturation of
oocytes has been demonstrated in rats by Mattheij, J. et al (1993),
Gynecol. Obstet. Invest. 36: 129-135]. On in vivo administration the
compounds of the invention specifically affects germ cells and therefore
have the advantage of maintenance of endogenous hormonal levels and
subsequently maintenance of normal cycle length. Such a contraceptive
method will not cause unwanted side-effects sometimes associated with
steroidal contraception (e.g. thrombosis, mood, unscheduled bleeding,
malignant breast disease). In this connection it is important to note that
the compounds of the invention do no bind to steroid receptors since no
binding was found for progesterone receptor, androgen receptor, estrogen
receptor and glucocorticoid receptor. Furthermore, it was found that
compounds did not have an effect on steroid synthesis or metabolism in
human adrenal cells at a dose level which induces oocyte maturation in
vitro.
A further advantage of the
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention is their inability to induce maturation in incompetent oocytes
(isolated from pre-antral follicles), which indicates that the compounds
will not affect the entire oocyte reserve in the ovaries. Only oocytes
from antral follicles (competent oocytes) can be induced to mature by the
compounds of the invention.
17.beta.-Aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention acting as inhibitors of the FF-Mas induced oocyte maturation can
be used for female contraception by inhibition of the naturally induced
oocyte maturation caused by the gonadotrophin surge. These compounds may
induce an ovulated not-matured oocyte which cannot be fertilized.
For treatment of female infertility caused by the absence of mature oocytes
the compounds of the invention can be administered in vivo to timely
stimulate the maturation of competent oocytes.
The compounds of the invention can also be used for suppletion of culture
media for in vitro fertilization procedures in order to improve oocyte
quality.
For male contraception the compounds of the invention can be administered
in vivo to inhibit the spermatogenesis.
For treatment of male infertility caused by a shortage of the number of
mature spermatozoa the compounds of the invention can be administered in
vivo to stimulate the maturation of spermatogonia.
The 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of this
invention have the natural configurations 5.alpha., 9.alpha., 10.beta.,
13.beta., 14.alpha., 17.beta., and possess also one or more additional
chiral carbon atoms. The compounds may therefore be obtained as a pure
diastereomer, or as a mixture of diastereomers. Methods for obtaining the
pure diastereomers are well known in the art, e.g. crystallization or
chromatography.
Compounds according to formula I wherein X is S(O) may exist as a
diastereoisomeric sulfoxide pair, due to the presence of the optically
active sulfur atom. Both the diastereoisomeric mixture and the separate
isomers are included in the present invention.
For therapeutic use, salts of the compounds of formula I are those wherein
the counterion is pharmaceutically acceptable. However, salts of the acids
according to formula I [i.e. compounds wherein R.sub.1 is (H,OSO.sub.3 H)]
may also find use, for example, in the preparation or purification of a
pharmaceutically acceptable compound. All salts, whether pharmaceutically
acceptable or not, are included within the ambit of the present invention.
Examples of salts of acids according to the invention are mineral salts
such as sodium salt, potassium salt, and salts derived from organic bases
like ammonia, imidazole, ethylenediamine, triethylamine and the like.
The compounds of formula I or a pharmaceutically acceptable salt thereof,
also referred to herein as the active ingredient, may be administered
enterally or parenterally. The exact dose and regimen of administration of
the active ingredient, or a pharmaceutical composition thereof, will
necessarily be dependent upon the therapeutic effect to be achieved
(treatment of infertility; contraception), and will vary with the
particular compound, the route of administration, and the age and
condition of the individual subject to whom the medicament is to be
administered. In general parenteral administration requires lower dosages
than other methods of administration which are more dependent upon
adsorption. However, a dosage for humans preferably contains 0.0001-25 mg
per kg body weight. The desired dose may be presented as one dose or as
multiple subdoses administered at appropriate intervals throughout the
day, or, in case of female recipients, as doses to be administered at
appropriate daily intervals throughout the menstrual cycle. The dosage as
well as the regimen of administration may differ between a female and a
male recipient.
In case of in vitro or ex vivo applications, like in IVF applications, the
compounds of the inventions are to be used in the incubation media in a
concentration of approximately 0.01-5 .mu.g/ml.
The present invention thus also relates to pharmaceutical compositions
comprising a 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative
according to formula I in admixture with pharmaceutically acceptable
auxiliaries, and optionally other therapeutic agents. The auxilliaries
must be "acceptable" in the sense of being compatible with the other
ingredients of the composition and not deleterious to the recipients
thereof.
Pharmaceutical compositions include those suitable for oral, rectal, nasal,
topical (including transdermal, buccal and sublingual), vaginal or
parenteral (including subcutaneous, intramuscular, intravenous and
intradermal) administration. The compositions may be prepared by any
method well known in the art of pharmacy, for example, using methods such
as those described in Gennaro et al., Remington's Pharmaceutical Sciences
(18th ed., Mack Publishing company, 1990, see especially Part 8:
Pharmaceutical Preparations and Their Manufacture). Such methods include
the step of bringing in association the active ingredient with any
auxilliary agent. The auxilliary agent(s), also named accessory
ingredients, include those conventional in the art (Gennaro, supra), such
as, fillers, binders, diluents, disintegrants, lubricants, colorants,
flavoring agents and wetting agents.
Pharmaceutical compositions suitable for oral administration may be
presented as discrete dosage units such as pills, tablets or capsules, or
as a powder or granules, or as a solution or suspension. The active
ingredient may also be presented as a bolus or paste. The compositions can
further be processed into a suppository or enema for rectal
administration.
For parenteral administration, suitable compositions include aqueous and
non-aqueous sterile injection. The compositions may be presented in
unit-dose or multi-dose containers, for example sealed vials and ampoules,
and may be stored in a freeze-dried (lyophilised) condition requiring only
the addition of sterile liquid carrier, for example, water prior to use.
Compositions, or formulations, suitable for administration by nasal
inhalation include fine dusts or mists which may be generated by means of
metered dose pressurised aerosols, nebulisers or insufflators.
The 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivatives of the
invention can also be administered in the form of implantable
pharmaceutical devices, consisting of a core of active material, encased
by a release rate-regulating membrane. Such implants are to be applied
subcutaneously or locally, and will release the active ingredient at an
approximately constant rate over relatively large periods of time, for
instance from weeks to years. Methods for the preparation of implantable
pharmaceutical devices as such are known in the art, for example as
described in European Patent 0,303,306 (AKZO N.V.).
The compounds of the invention may be produced by various methods known in
the art of organic chemistry in general, and especially in the art of the
chemistry of steroids (see, for example: Fried, J. and Edwards, J. A.,
"Organic Reactions in Steroid Chemistry", Volumes I and II, Van Nostrand
Reinhold Company, New York, 1972). A suitable method for the preparation
of compounds of formula I is the condensation of a compound of general
formula II,
##STR3##
wherein R.sub.2 and R.sub.3 are independently hydrogen or (C.sub.1-6)alkyl,
R.sub.9 is a hydroxy-protecting group such as an acyl group, like an
acetyl group, a benzoyl group or a pivaloyl group, or an alkoxyalkyl
group, like an ethoxyethyl group or a tetrahydropyranyl (THP) group, n is
0, 1 or 2, and wherein the dotted lines represent a .DELTA..sup.7 or a
.DELTA..sup.8 double bond, or a pair of conjugated double bonds selected
from .DELTA..sup.7,14, .DELTA..sup.8,14, or .DELTA..sup.6,8(14), and
wherein Y is OH, with a (thio)phenol derivative or benzyl
alcohol(mercaptan) derivative having formula III, wherein R.sub.4,
R.sub.5, and R.sub.6 have the previously given meaning, Z is OH or SH and
m is 0 or 1, or the reaction of a compound according to formula II wherein
Y is a halogen, preferably bromide, or a leaving group like tosyloxy or
mesyloxy, with a compound according to formula III wherein Z is OH or SH,
or the reaction of a compound according to formula II wherein Y is OH or
SH with a compound according to formula III (m=1) wherein Z is a halogen,
preferably bromide, or a leaving group like tosyloxy or mesyloxy,
whereafter a 17-aryl(arylmethyl)thioalkyl-androstane derivative is
optionally oxidized to the corresponding sulfoxide (X=SO) or sulfone
(X=SO.sub.2) derivative, after which any remaining protecting groups are
removed, and the 3-OH group of the product is optionally converted to a
3-OR group, wherein R has the meaning as previously defined, or wherein
the 3-OH group is oxidized and the resulting 3-keto-group converted to
NOR, wherein R has the meaning as previously defined.
The condensation reactions between compounds having formula II and
compounds having formula lIl are carried out using methods known in the
art. For instance, compounds of formula I (X=O,S; n=0,1,2; m=0) can be
prepared by means of a Mitsunobu reaction between a compound of formula II
wherein Y is OH with an unsubstituted or suitably substituted phenol or
thiophenol derivative [a compound having formula III (m=0)] [see Hughes,
D. L., Organic Reactions 42, 335 (1992)]. This reaction can be carried out
using a dialkyl azodicarboxylate, like diethyl azodicarboxylate or
diisopropyl azodicarboxylate, or other azodicarbonyl derivatives, like
1,1'-(azodicarbonyl)dipiperidine [Tsunoda, T. et al, Tetrahedron Lett. 34,
1639 (1993)]. Phosphines which can be used are: triarylphosphines like
triphenylphosphine, trialkylphosphines like tributylphosphine, or
trialkylphosphites like trimethylphosphite.
The condensation reaction can also be carried out by means of an
etherification reaction, i.e. by reaction of compounds of general formula
II, wherein Y is a leaving group such as bromide, iodide, tosyloxy and
mesyloxy, with a suitably substituted phenol or thiophenol derivative,
which is previously converted to the lithium, sodium or potassium salt
[see e.g. Meerwein, H., Methoden der Organischen Chemie (Houben-Weyl),
Band VI/3, p. 1, Georg Thieme Publishers, Stuttgart, 1965; Yin, J. et al,
Tetrahedron Lett. 38, 5953 (1997) and references cited therein]. Compounds
of formula I (X=O,S; n=0,1,2; m=1) can be prepared analogously by means of
an etherification reaction between a compound of formula II wherein Y is a
leaving group such as bromide, iodide, tosyloxy and mesyloxy, with a
suitably substituted benzyl alcohol or benzyl marcaptan derivative, which
is previously converted to the lithium, sodium or potassium salt. In this
case, the etherification reaction can also be carried out in the opposite
direction, i.e. by reaction between a metallated (Li, Na, K) compound of
general formula II (Y=OH,SH) and a compound of formula IIII (m=1) in which
Z is a leaving group such as bromide, iodide, tosyloxy and mesyloxy.
Compounds of general formula I wherein X is S(O) or S(O).sub.2 can be
prepared from compounds of formula I wherein X is S by oxidation using
agents capable of converting a thio ether into a sulfoxide or a sulfone,
e.g. H.sub.2 O.sub.2 and the like [see Varma, R. S. et al, Tetrahedron
Lett. 38, 6525 (1997) and references cited therein]. Compounds of general
formula I (X=S(O).sub.2 ; n=1,2; m=0) can also be prepared directly by
reaction of compounds of general formula II in which Y is a leaving group
such as bromide, iodide, tosyloxy and mesyloxy, with an unsubstituted or
suitably substituted benzenesulfinic acid derivative, which is previously
converted to the sodium or potassium salt [see Trost, B. M. et al, J. Am.
Chem. Soc. 105, 5075 (1983)].
A convenient starting material for the preparation of compounds having
formula II (n=0) is (3.beta.)-3-hydroxypregn-5-en-20-one (compound 1; see
Scheme 1) which is commercially available. A possible synthesis route
(specific examples are exemplified in Example 1 and 2, and Scheme 1 and 2)
starts with the protection of the hydroxy group at C-3 by an ether type
protecting group, e.g. an ethoxyethyl ether or a THP ether, or by a silyl
ether. These and other suitable protective groups are known in the art,
e.g. from Greene, T. W. and Wuts, P. G. M.: Protective groups in Organic
Synthesis, Wiley, New York, 1991.
In a second step, the carbonyl function at C-20 is reduced to a hydroxy
group. The reduction can be carried out using hydride reducing agents,
like lithium aluminium hydride, sodium borohydride, and the like, in which
case the 20R isomer is mainly formed [Antia, N. J. et al, J. Chem. Soc.
1218 (1954)]. The reduction can also be carried out using alkalimetals in
alkanols [Meystre, C. Helv. Chim. Acta 29, 33 (1946)] or with boranes
[Midland, M. M. et al, J. Amer. Chem. Soc. 105, 3725 (1983)], which lead
to the predominant formation of the 20S isomer.
As an alternative the 3-protected pregn-5-ene-3,20-diols can be prepared by
hydroboration of suitably protected pregna-5, 17(20)-dien-3-ol derivatives
[Murayama, E. et al, Chem. Pharm. Bull. 34, 4410 (1986); Yoshizawa, I. et
al, Chem. Pharm. Bull. 31, 3819 (1983)]. The hydroxy group at C-20 is
converted to an ester, e.g. an acetate ester or a benzoate ester and the
like and the 3-hydroxy function is deprotected. The latter is oxidized to
a carbonyl group, which can be accomplished by an Oppenauer oxidation or a
Swern oxidation, or by using chromium(VI) reagents, e.g. Jones reagent,
pyridinium dichromate, pyridium chlorochromate or other oxidizing agents
known in the art. The hydroxy group at C-20 is deprotected by
saponification and then reprotected as a silyl ether, e.g. a
diisopropylsilyl ether, a t-butyldimethylsilyl ether, or a
t-butyldiphenylsilyl ether.
Optionally, the resulting product can now be mono- or dialkylated at C-4,
for instance, it can be dialkylated with methyl. Alkylation can be
performed using standard procedures [such as potassium
t-butoxide/methyliodide in t-butanol/-tetrahydrofuran: Dolle, R. E. et al,
J. Org. Chem. 51, 4047 (1986)], it can also be accomplished by other
techniques, e.g. lithium diisopropylamide/methyliodide in tetrahydrofuran,
and similar methods known in the art. Alternatively, the .DELTA..sup.4
compound can be converted to a .DELTA..sup.5 derivative by reaction with a
base followed by quenching with water [Jones, J. B. et al, Can. J. Chem.
46, 1459 (1968)]. In both cases, the carbonyl group at C-3 is reduced to
hydroxy. Reducing agents which can be used include lithium aluminium
hydride, sodium borohydride, or other hydride reducing agents known in the
art. The resulting 3-hydroxy compound is protected as an ester, e.g. an
acetate ester, a benzoate ester, or a pivalate ester and the like.
The .DELTA..sup.5 system can now be converted to a .DELTA..sup.5,7 -diene
system by the sequence: bromination at C-7 followed by dehydrobromination.
The bromination reaction can be carried out thermally [Schroepfer, G. J.,
Jr., et al, Chem. Phys. Lipids 47, 187 (1988)] or photochemically [Prelle,
A. et al, Heterocycles 28, 333 (1989)]. In both cases, brominating agents
which can be used are N-bromosuccinimide,
1,3-dibromo-5,5-dimethylhydantoin and the like. Dehydrobrominating agents
include N,N-diisopropylethylamine, 2,4,6-trimethylpyridine,
trimethylphosphite, tetrabutylammonium fluoride and others.
The .DELTA..sup.5,7 diene system can be converted to a .DELTA..sup.7,14
diene system, a .DELTA..sup.8,14 diene system, or a .DELTA..sup.6,8(14)
diene system. Methods used are known in the art. For conversion to the
.DELTA..sup.7,14 derivative, see e.g. Wilson, W. K. et al, J. Org. Chem.
53, 1713 (1988). For conversion to the .DELTA..sup.8,14 derivative, see
e.g. Schroepfer, G. J., Jr., et al, Chem. Phys. Lipids 47, 187 (1988) or
Dolle, R. E. et al, J. Org. Chem. 53, 1563 (1988). For conversion to the
.DELTA..sup.6,8(14) derivative, see e.g. Kaneko, C. et al, Chem. Pharm.
Bull. 26, 3582 (1978). .DELTA..sup.7 Compounds are obtained from the
.DELTA..sup.5,7 diene system by reduction with lithium in liquid ammonia
[Lederer, F. et al, Bull. Soc. Chim. Fr. 1295 (1965)] or by hydrogenation.
Hydrogenation catalysts which can be used include Raney nickel [Gautschi,
F. et al, J. Biol. Chem. 233, 1343 (1958)], Wilkinson's catalyst
[Canonica, L. et al, Steroids 11, 287 (1968)] and others. .DELTA..sup.8
Derivatives are prepared from .DELTA..sup.8,14 dienes by the sequence:
selective hydroboration of the .DELTA..sup.14 double bond followed by
deoxygenation of the 15-hydroxy compound produced [Dolle, R. E. et al, J.
Amer. Chem. Soc. 111, 278 (1989)].
Deprotection of the hydroxy group at C-20 (if necessary, in some cases
manipulation of the .DELTA..sup.5,7 system can be accompanied by
deprotection of that group) results in the unsaturated pregna-3,20-diol
derivatives of formula II (Y=OH; n=0) in which the 3-hydroxy function is
protected as an ester (R.sub.9 =acyl) (e.g. compound 13a; Scheme 1).
Unsaturated pregna-3,20-diol derivatives of formula II (Y=OH; n=0) in which
the 3-hydroxy function is protected as an alkoxyalkyl ether (e.g. compound
19b; Scheme 2) can be obtained from compounds of formula II
[Y=(protected)OH; n=0; R.sub.9 =acyl] as follows: the 20-hydroxy function
is protected as a silyl ether as described before (if necessary), the
ester function at C-3 is removed by reduction with lithium aluminium
hydride, or by other hydride reducing agents known in the art, whereafter
the 3-hydroxy group is reprotected as an alkoxyalkyl ether, e.g. an
ethoxyethyl ether or a tetrahydropyranyl ether. Finally, treatment with a
fluoride agent, e.g. potassium fluoride, tetrabutylammonium fluoride or
other reagents known in the art then results in the formation of the
unsaturated pregna-3,20-diol derivatives of formula II (Y=OH; n=0) in
which the 3-hydroxy function is protected as an alkoxyalkyl ether.
Compounds having formula II (Y=OH; n=1) can be obtained from e.g.
(20S)-21-hydroxy-20-methylpregn-4-en-3-one [Trost, B. M. et al, J. Amer.
Chem. Soc. 105, 5075 (1983)] by a sequence of reactions analogous to that
described above.
Compounds having formula II (Y=OH; n=2) can be prepared from compounds of
formula II (Y=OH; n=1) by homologation, e.g. by the reaction sequence:
conversion of the 21-hydroxy group to a leaving group, reaction with
potassium cyanide, reduction of the carbonitrile group to the
corresponding carboxaldehyde group, reprotection of the 3-hydroxy group as
an alkoxyalkyl ether, like an ethoxyethyl group or a THP group and,
finally, reduction to the 23-hydroxy derivative. As an alternative a
one-carbon homologation starting with the corresponding 20-carboxaldehyde
can be accomplished via a Wittig condensation with (Ph).sub.3 P.dbd.CHOMe
and hydrolysis of the intermediate enol ether. Techniques for homologation
are well known in the art, see e.g. Mathieu, J. et al: Formation of C--C
Bonds, Vol. I-III, Georg Thieme Publishers, Stuttgart, 1973.
Compounds having formula II (Y=SH; n=0,1,2) can be prepared from compounds
of formula II (Y=OH; n=0,1,2) by means of a Mitsunobu reaction with
thioacetic acid followed by conversion to the thiol derivative by
reduction or saponification [see Hughes, D. L., Organic Reactions 42, 335
(1992)]. Alternatively, they can be prepared by reaction of compounds of
general formula II in which Y is a leaving group such as bromide, iodide,
tosyloxy and mesyloxy, with thiourea followed by reaction with a base,
like sodium hydroxide or potassium hydroxide [see e.g. Allewaert, K. et
al, Bioorganic & Med. Chem. Lett. 3, 1859 (1993)].
Compounds of formula I in which R.sub.1 is (H,OH) may serve as starting
material for the synthesis, using methods known in the art, of compounds
of formula I in which R.sub.1 is (H,OR), (H,OSO.sub.3 H) or NOR, and R is
H, (C.sub.1-6)alkyl, or (C.sub.1-6)acyl.
The invention is further illustrated with reference to the following
schemes and examples.
##STR4##
##STR5##
##STR6##
i: Ethyl vinyl ether, PPTS, CH.sub.2 Cl.sub.2. ii: Na, n-butanol, .DELTA.;
3a:3b=59:41. iii: Ac.sub.2 O, pyridine. iv: HCl, acetone. v:
Al(i-OPr).sub.3, 2-butanone, toluene, .DELTA.. vi: KOH, MeOH, THF, H.sub.2
O. vii: t-Butyldimethylsilyl chloride (TBSCl), imidazole, DMF. viii: Mel,
t-BuOK, t-BuOH, THF, 45.degree. C. ix: LiAlH.sub.4, THF. x: BzCl,
pyridine. xi: 1) NBS, cyclohexane, toluene, .DELTA.. 2) EtN(i-Pr).sub.2,
toluene, .DELTA.. xii: HCl, H.sub.2 O, EtOH, toluene, .DELTA.. xiii:
1,1'-(Azodicarbonyl)dipiperidine, triphenylphosphine, phenol, THF. xiv:
LiAlH.sub.4, THF.
##STR7##
##STR8##
i: t-Butyldimethylsilyl chloride (TBSCl), imidazole, DMF. ii: LiAlH.sub.4,
THF. iii: Ethyl vinyl ether, PPTS, CH.sub.2 Cl.sub.2. iv: TBAF, THF. v:
C.sub.6 H.sub.5 CH.sub.2 Br, t-BuOK, 18-crown-6, toluene, .DELTA.. vi:
HCl, acetone.
EXAMPLE 1
(3.beta.,5.alpha.,20R)-4,4-Dimethyl-20-phenoxypregna-8,14-dien-3-ol (15b;
Scheme I)
i)--Pyridium p-toluenesulfonate (6.32 g) was added to a solution of
(3.beta.)-3-hydroxypregn-5-en-20-one (1; 100 g) in dichloromethane (600
ml) and ethyl vinyl ether (200 ml). After stirring of the reaction mixture
for 1 h at room temperature triethylamine (20 ml) was added, whereupon the
mixture was poured into a saturated aqueous solution of sodium hydrogen
carbonate (1 l). The product was extracted into dichloromethane; the
combined organic phases were washed with brine, dried over sodium sulfate,
and concentrated under reduced pressure to give
(3.beta.)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-one (2; 125.6 g), which was
used in the following step without further purification.
ii)--Sodium (8.98 g) was added in small portions in the course of 2 h to a
refluxing solution of (3.beta.)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-one
(20 g) in dry n-butanol (587 ml). Heating was continued for another 2 h;
then the reaction mixture was cooled and poured into a saturated aqueous
solution of sodium hydrogen carbonate (1.5 l). The product was extracted
into ethyl acetate; the combined organic phases were washed with brine,
dried over sodium sulfate, and concentrated under reduced pressure. Column
chromatography of the crude product afforded
(3.beta.,20R)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-ol (compound 3b; 5.08 g)
and (3.beta.,20S)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-ol (compound 3a;
7.21 g).
iii)--A solution of (3.beta.,20S)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-ol
(compound 3a; 56.9 g) in a mixture of dry pyridine (144 ml) and acetic
anhydride (72.4 ml) was stirred at room temperature for 18 h. Water (482
ml) was added and stirring was continued for another 1 h. The product was
extracted into ethyl acetate; the combined organic phases were washed with
a saturated aqueous solution of sodium hydrogen carbonate and with brine,
dried over sodium sulfate, and concentrated under reduced pressure to give
(3.beta.,20S)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-ol acetate (compound 4a;
66.1 g) which was used in the following step without further purification.
iv)--A solution of (3.beta.,20S)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-ol
acetate (66.1 g) in acetone (1330 ml) was treated with a 4 M aqueous
solution of hydrochloric acid (13.2 ml). The reaction mixture was stirred
for 45 minutes and subsequently poured into a mixture of water (3 l) and a
saturated aqueous solution of sodium hydrogen carbonate (500 ml). The
resulting precipitate was collected by filtration and then dissolved in
dichloromethane. The filtrate was extracted with ethyl acetate. The
dichloromethane and ethyl acetate solutions were washed with a saturated
aqueous solution of sodium hydrogen carbonate and with brine, dried over
sodium sulfate, and concentrated under reduced pressure to give
(3.beta.,20S)-pregn-5-ene-3,20-diol 20-acetate (compound 5a; 53.5 g),
which was used in the following step without further purification.
v)--Aluminium isopropoxide (18.1 g) was added to a solution of
(3.beta.,20S)-pregn-5-ene-3,20-diol 20-acetate (21.9 g) in dry toluene
(252 ml) and dry 2-butanone (156 ml). The mixture was heated under reflux
for 2 hours, then cooled, whereupon a solution of potassium sodium
tartrate tetrahydrate (91.4 g) in water (90 ml) was added. The mixture was
stirred for 30 min. and filtered. The filtrate was poured into brine and
the product extracted into ethyl acetate. The combined organic phases were
washed with brine, dried over sodium sulfate, and concentrated under
reduced pressure. Column chromatography afforded
(20S)-20-(acetyloxy)pregn-4-en-3-one (compound 6a; 17.9 g).
vi)--Potassium hydroxide (22.5 g) was added to a solution of
(20S)-20-(acetyloxy)pregn-4-en-3-one (17.9 g) in tetrahydrofuran (418 ml),
methanol (380 ml) and water (125 ml). The mixture was stirred for 3 h at
room temperature and then poured into water (2 l). The product was
extracted into ethyl acetate; the combined organic phases were washed with
brine, dried over sodium sulfate, and concentrated under reduced pressure.
Column chromatography afforded (20S)-20-hydroxypregn-4-en-3-one (compound
7a; 35.4 g).
vii)--t-Butyldimethylsilyl chloride (39.6 g) was added to a solution of
(20S)-20-hydroxypregn-4-en-3-one (36.3 g) and imidazole (53.5 g) in dry
N,N-dimethylformamide (345 ml). The reaction mixture was stirred for 2.5
hours at room temperature and then poured into water. The product was
extracted into ethyl acetate. The combined organic phases were washed with
water and brine and dried over sodium sulfate. Column chromatography
afforded (20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]pregn-4-en-3-one
(compound 8a; 41.6 g).
viii)--Potassium t-butoxide (46.3) was added to a solution of
(20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]pregn-4-en-3-one (40.6 g)
in dry t-butanol (1000 ml) and dry tetrahydrofuran (146 ml). After heating
the mixture at 45.degree. C. for 10 min., iodomethane (52.4 ml) was added.
The reaction mixture was heated at the same temperature overnight and then
poured into water (2 l). The product was extracted into ethyl acetate; the
combined organic phases were washed with brine and dried over sodium
sulfate. Column chromatography afforded
(20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dimethylpregn-5-en-3-
one (compound 9a; 40.7 g).
ix)--A solution of compound 9a (39.4 g) in dry tetrahydrofuran (400 ml) was
added dropwise to an ice-cooled suspension of lithium aluminium hydride
(9.81 g) in tetrahydrofuran (490 ml). The mixture was stirred at room
temperature for 1 h. The reaction was cooled to 0.degree. C., and then
quenched with a saturated aqueous solution of sodium sulfate. The reaction
mixture was filtered over celite and the filtrate concentrated under
reduced pressure to give
(3.beta.,20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dimethylpregn
-5-en-3-ol (compound 10a; 38.9 g), which was used in the following step
without further purification.
x)--Benzoyl chloride (19.9 ml) was added in 5 min. to an ice-cooled
solution of
(3.beta.,20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dimethylpregn
-5-en-3-ol (38.9 9) in dry pyridine (395 ml). The reaction mixture was
stirred for 2 h at room temperature and then poured into ice-water (2 l).
The resulting suspension was stirred overnight and filtered; the residue
was washed with water of 40-50.degree. C. (1.5 l) and then dissolved in
dichloromethane. The dichloromethane solution was washed with brine, dried
over sodium sulfate, and concentrated under reduced pressure. Column
chromatography afforded
(3.beta.,20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dimethylpregn
-5-en-3-ol benzoate (compound 11a; 45.9 g).
xi)--A mixture of
(3.beta.,20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dimethylpregn
-5-en-3-ol benzoate (44.6), dry toluene (394 ml), dry cyclohexane (394 ml)
and N-bromosuccinimide (17.8 g) was heated at reflux temperature for 10
min. The reaction mixture was cooled, another portion of
N-bromosuccinimide (17.8 g) was added, and reflux was continued for
another 10 min. The reaction mixture was cooled, a saturated aqueous
solution of sodium thiosulfate (802 ml) was added and the resulting
mixture was stirred for 30 min. The organic phase and the aqueous phase
were separated and the latter extracted two times with toluene. The
combined organic phases were dried over sodium sulfate and concentrated
under reduced pressure. A solution of the crude product thus obtained in
dry toluene (1381 ml) and N,N-diisopropylethylamine (138 ml) was heated
under reflux for 1.5 h. Then it was cooled and washed with a saturated
aqueous solution of sodium hydrogen carbonate, a saturated aqueous
solution of ammonium chloride and with brine, the aqueous phase each time
being extracted with ethyl acetate. The combined toluene and ethyl acetate
solutions were dried over sodium sulfate and concentrated under reduced
pressure. Column chromatography afforded
(3.beta.,20S)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dimethylpregn
a-5,7-dien-3-ol benzoate (compound 12a; 40.0 g).
xii)--A mixture of compound 12a (36.6 g), toluene (91 ml), ethanol (622 ml;
96%) and concentrated hydrochloric acid (91 ml) was heated under reflux
for 4 h. The mixture was cooled and poured into a saturated aqueous
solution of sodium hydrogen carbonate (1 l). The product was extracted
into ethyl acetate, the combined organic phases were washed with a
saturated aqueous solution of sodium hydrogen carbonate and with brine,
dried over sodium sulfate, and concentrated under reduced pressure. Column
chromatography and crystallization afforded
(3.beta.,5.alpha.,20S)-4,4-dimethylpregna-8,14-diene-3,20-diol 3-benzoate
(compound 13a; 7.89 g) and
(3.beta.,5.alpha.,20S)-4,4-dimethylpregna-6,8(14)-diene-3,20-diol
3-benzoate (3.69 g).
xiii)--1,1'-(Azodicarbonyl)dipiperidine (5.54 g) was added to an ice-cooled
solution of compound 13a (2.0 g), triphenylphosphine (5.76 g), and phenol
(2.06 g) in dry THF (10 ml). After 5 min. stirring dry THF (60 ml) was
added and the reaction mixture was stirred for 2 h at room temperature.
Then it was poured into heptane (100 ml); the resulting mixture was
filtered and the filtrate was concentrated under reduced pressure. Column
chromatography afforded
(3.beta.,5.alpha.,20R)4,4-dimethyl-20-phenoxypregna-8,14-dien-3-ol
benzoate (compound 14b), together with the elimination products
(3.beta.,5.alpha.)-4,4-dimethylpregna-8,14,17(20)-trien-3-ol benzoate and
(3.beta.,5.alpha.)-4,4-dimethylpregna-8,14,20-trien-3-ol benzoate (2.80 g
totally). The product was used in the following step without further
purification.
xiv)--Following a procedure analogous to that of step ix, the products of
the previous step (2.80 g) were converted to the 3-hydroxy compounds to
give, after column chromatography and crystallization,
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-phenoxypregna8,14-dien-3-ol
(compound 15b, 0.049 g). M.p. 135-140.degree. C.; [.alpha.].sub.D.sup.20
=-30.4.degree. (c=0.5, dioxane).
EXAMPLE 2
(3.beta.,5.alpha.,20R)-4,4-Dimethyl-20-(phenylmethoxy)pregna-8,14-dien-3-ol
(21b; Scheme 2)
Starting from
(3.beta.,5.alpha.,20R)-4,4-dimethylpregna-8,14-diene-3,20-diol 3-benzoate
(compound 13b), which was prepared from
(3.beta.,20R)-3-[(1-ethoxyethyl)oxy]pregn-5-en-20-ol (compound 3b; Example
1, step ii) using reaction steps analogous to those described under
iii--xii of Example 1 for
(3.beta.,5.alpha.,20S)-4,4-dimethylpregna-8,14-diene-3,20-diol 3-benzoate
(compound 13a), the title compound was prepared as follows:
i)--Following a procedure analogous to that of step vii of Example 1,
(3.beta.,5.alpha.,20R)-4,4-dimethylpregna-8,14-diene-3,20-diol 3-benzoate
(compound 13b, 15.2 g) was converted to
(3.beta.,5.alpha.,20R)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dime
thylpregna-8,14-dien-3-ol benzoate (compound 16b; 23.1 g).
ii)--Following a procedure analogous to that of step ix of Example 1,
(3.beta.,5.alpha.,20R)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4-dime
thylpregna-8,14-dien-3-ol benzoate (23.1 g) was converted to
(3.beta.,5.alpha.,20R)-20-[[(1,1-dimethylethyl)dimethyl-silyl]oxy]-4,4-dim
ethylpregna-8,14-dien-3-ol (compound 17b; 19.7 g).
iii)--Following a procedure analogous to that of step i of Example 1
compound 17b (15.3 g) was converted to
(3.beta.,5.alpha.,20R)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-3-[(1-et
hoxyethyl)oxy]-4,4-dimethylpregna-8,14-diene (compound 18b; 16.4 g).
iv)--A solution of
(3.beta.,5.alpha.,20R)-20-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-3-[(1-et
hoxyethyl)oxy]-4,4-dimethylpregna-8,14-diene (18b; 16.4 g) in a 1 M
solution of tetrabutylammonium fluoride in tetrahydrofuran (42 ml) was
stirred at 50.degree. C. for 6 h and then at room temperature for 24 h.
The reaction mixture was poured into water and the product was extracted
into ethyl acetate. The combined organic phases were washed with brine and
concentrated under reduced pressure. Column chromatography afforded
(3.beta.,5.alpha.,20R)-3-[(1-ethoxyethyl)oxy]-4,4-dimethylpregna-8,14-dien
-20-ol (compound 19b; 10.6 g).
v)--A mixture of
(3.beta.,5.alpha.,20R)-3-[(1-ethoxyethyl)oxy]-4,4-dimethylpregna-8,14-dien
-20-ol (19b; 0.80 g), dry toluene (56 ml), potassium t-butoxide (2.76 g),
18-crown-6 (0.48 g) and benzyl bromide (1.13 ml) was heated at 70.degree.
C. for 1 h. After cooling, the reaction mixture was poured into water and
the product extracted into ethyl acetate. The combined organic phases were
washed with water and with brine, dried over sodium sulfate, and
concentrated under reduced pressure to give
(3.beta.,5.alpha.,20R)-3-[(1-ethoxyethyl)oxy]-4,4-dimethyl-20-(phenylmetho
xy)pregna-8,14-diene (compound 20b; 1.92 g), which was used in the
following step without further purification.
vi)--Following a procedure analogous to that of step iv of Example 1,
(3.beta.,5.alpha.,20R)-3-[(1-ethoxyethyl)oxy]-4,4-dimethyl-20-(phenylmetho
xy)pregna-8,14-diene (compound 20b; 1.92 g), was converted to
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-(phenylmethoxy)pregna-8,14-dien-3-o
l (compound 21b; 0.54 g). M.p. 138-143.degree. C.; [.alpha.].sub.D.sup.20
=-46.8.degree. (c=0.5, dioxane).
EXAMPLE 3
Starting from
(3.beta.,5.alpha.,20R)-3-[(1-ethoxyethyl)oxy]-4,4-dimethylpregna-8,14-dien
-20-ol (compound 19b) described under iv of Example 2, and using reaction
steps analogous to those described under v and vi of Example 2, the
following compounds were prepared:
A)
(3.beta.,5.alpha.,20R)-20-[(2-Fluorophenyl)methoxy]-4,4-dimethylpregna-8,1
4-dien-3-ol. M.p. 149-151.degree. C.
B)
(3.beta.,5.alpha.,20R)-20-[(3-Fluorophenyl)methoxy]-4,4-dimethylpregna-8,1
4-dien-3-ol. M.p. 113-115.degree. C.
C)
(3.beta.,5.alpha.,20R)-20-[(4-Fluorophenyl)methoxy]-4,4-dimethylpregna-8,1
4-dien-3-ol. M.p.104-106.degree. C.
D)
(3.beta.,5.alpha.,20R)-20-[(3,4-Difluorophenyl)methoxy]-4,4-dimethylpregna
-8,14-dien-3-ol. M.p. 48-50.degree. C.
E)
(3.beta.,5.alpha.,20R)-4,4-Dimethyl-20-[[2-(trifluoromethyl)phenyl]methoxy
]pregna-8,14-dien-3-ol. M.p. 65-67.degree. C. The product contained 20% of
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-[[2-(trifluoromethyl)phenyl]methoxy
]pregna-6,8(14)-dien-3-ol.
EXAMPLE 4
(3.beta.,5.alpha.,20S)-4,4,20-Trimethyl-21-phenoxypregna-8,14-dien-3-ol
i)--A solution of (20S)-3-oxopregn-4-ene-20-carboxaldehyde (125 g) in dry
ethanol (1250 ml) was cooled to -10.degree. C., whereupon a solution of
sodium borohydride (4.4 g) in dry ethanol (80 ml) was added in 30 min.
After stirring the mixture for 2 hours at -10.degree. C., the reaction was
quenched by adding a 50% aqueous solution of acetic acid. The reaction
mixture was concentrated under reduced pressure to 25% of its original
volume and then poured into ice-water (5 l). The resulting suspension was
stirred overnight and filtered. The residue was washed with water and
dried to give (20S)-21-hydroxy-20-methylpregn-4-en-3-one (124 g) which was
used in the following step without further purification.
ii)--A solution of the alcohol (124 g) obtained in the previous step and of
imidazole (176 g) in dry N,N-dimethylformamide (1730 ml) was cooled to
10.degree. C. t-Butyldimethylsilyl chloride (112 g) was added in one
portion and the mixture was stirred at room temperature for 2 h. Then it
was poured into a mixture of ice-water (10 l) and of a saturated aqueous
solution of sodium hydrogen carbonate (750 ml). The resulting suspension
was filtered and the residue was washed with water. Drying of the residue
afforded
(20S)-21-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-20-methylpregn-4-en-3-one
(169.3 g), which was used in the following step without further
purification.
iii)--A mixture of potassium t-butoxide (169.5 g) and dry t-butanol (3750
ml) was warmed to 45.degree. C. A solution of the ketone obtained in the
previous step (169.3 g) in dry tetrahydrofuran (375 ml) was added and the
mixture was stirred for 10 min. Iodomethane (187.5 ml) was added in 10
min. and stirrring was continued for 3 h. The reaction mixture was
concentrated under reduced pressure to a volume of 1.5 l and then poured
into ice-water (10 l). After stirring of the mixture for 2 hours the
suspension was filtered. The residue was washed with water, dried and
purified by crystallization from acetone to give
(20S)-21-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4,20-trimethylpregn-5-e
n-3-one (136.9 g).
iv)--A solution of the ketone (140 g) obtained in the previous step in dry
tetrahydrofuran (1400 ml) was added in 30 min. to an ice-cooled suspension
of lithium aluminium hydride (35 g) in tetrahydrofuran (1750 ml). After
stirring of the mixture for 1 hour at room temperature, the reaction was
quenched by addition of a saturated aqueous solution of sodium sulfate
(152 ml), followed by water (39 ml). Ethyl acetate (1750 ml) was added,
and the mixture was filtered over celite. The filtrate was concentrated
under reduced pressure to give
(3.beta.,20S)-21-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4,20-trimethylp
regn-5-en-3-ol (136.3 g), which was used in the following step without
further purification.
v)--A solution of the alcohol (132.5 g) obtained in the previous step in
dry pyridine (1310 ml) was cooled to 0.degree. C. Benzoyl chloride (65.7
ml) was added in 5 min. and the reaction mixture was stirred for 1 h at
room temperature. Then it was poured into ice-water (6650 ml) and the
resulting suspension was stirred overnight. The precipitate was collected
by filtration and washed with water (40-50.degree. C.). The residue was
dried and purified by crystallization from acetone to give
(3.beta.,20S)-21-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4,20-trimethylp
regn-5-en-3-ol benzoate (113.6 g).
vi)--A mixture of the benzoate (94.0 g) obtained in the previous step, dry
toluene (810 ml), dry cyclohexane (810 ml) and N-bromosuccinimide (36.1 g)
was heated under reflux for 10 min. The reaction mixture was cooled,
another portion of N-bromosuccinimide (36.1 g) was added, and reflux was
continued for another 10 min. The reaction mixture was cooled, a saturated
aqueous solution of sodium thiosulfate (1620 ml) was added and the
resulting mixture was stirred for 30 min. The organic phase and the
aqueous phase were separated and the latter extracted two times with
toluene. The combined organic phases were dried over sodium sulfate and
concentrated under reduced pressure. A solution of the crude product thus
obtained in dry toluene (2835 ml) and N,N-diisopropylethylamine (284 ml)
was heated under reflux for 1 h. Then it was cooled and washed with a
saturated aqueous solution of sodium hydrogen carbonate, a saturated
aqueous solution of ammonium chloride and with brine, the aqueous phase
each time being extracted with ethyl acetate. The combined toluene and
ethyl acetate solutions were dried over sodium sulfate and concentrated
under reduced pressure to give
(3.beta.,20S)-21-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4,20-trimethylp
regna-5,7-dien-3-ol benzoate (134 g) which was used in the following step
without further purification.
vii)--A mixture of the diene obtained in the previous step (30.9 g),
chloroform (300 ml), and a solution of HCl in acetic acid (1 M, 300 ml)
was stirred for 45 min. at room temperature and then heated under reflux
for another 45 min. After cooling, the mixture was poured into water and
the product extracted into dichloromethane. The combined organic phases
were washed with water, a saturated aqueous solution of sodium hydrogen
carbonate and brine, dried over sodium sulfate and concentrated under
reduced pressure. Column chromatography afforded a 5:1 mixture (10.2 g) of
(3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-8,14-diene-3,21-diol
21-acetate 3-benzoate and
(3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-6,8(14)-diene-3,21-diol
21-acetate 3-benzoate. The mixture was used as such in the next step.
viii)--Following a procedure analogous to that described for step vi of
Example 1, the products of the previous step (10.2 g) were converted to
the 21-hydroxy derivatives. Column chromatography afforded a 5:1 mixture
(8.23 g) of
(3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-8,14-diene-3,21-diol
3-benzoate and
(3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-6,8(14)-diene-3,21-diol
3-benzoate which was used as such in the following step.
ix)--Following a procedure analogous to that described for step xiii of
Example 1, the products of the previous step (3.07 g) were converted to
the phenoxy compounds. Column chromatography afforded a 5:1 mixture (3.94
g) of
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-phenoxypregna-8,14-dien-3-ol
benzoate and
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-phenoxypregna-6,8(14)-dien-3-ol
benzoate which was used as such in the following step.
x)--Following a procedure analogous to that described for step ix of
Example 1, the products of the previous step (3.94 g) were converted to
the 3-hydroxy compounds. Column chromatography afforded a 5:1 mixture
(1.79 g) of
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-phenoxypregna-8,14-dien-3-ol
and
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-phenoxypregna-6,8(14)-dien-3-ol
which could be separated from each other by preparative HPLC (column:
Waters Symmetry C.sub.18, 19.times.150 mm; eluent: acetonitrile/water
4:1).
(3.beta.,5.alpha.,20S)-4,4,20-Trimethyl-21-phenoxypregna-8,14-dien-3-ol.
M.p. 134-137.degree. C.
EXAMPLE 5
Starting from the mixture of
(3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-8,14-diene-3,21-diol
3-benzoate and
(3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-6,8(14)-diene-3,21-diol
3-benzoate, described under viii of Example 4, and using reaction steps
analogous to those described under ix and x of Example 4, the following
compounds were prepared:
A)
(3.beta.,5.alpha.,20S)-4,4,20-Trimethyl-21-(2-methylphenoxy)pregna-8,14-di
en-3-ol. M.p. 126-141.degree. C.; [.alpha.].sub.D.sup.20 =-50.degree.
(c=0.5, dioxane). The product contained 15% of
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(2-methylphenoxy)pregna-6,8(14)
-dien-3-ol.
B)
(3.beta.,5.alpha.,20S)-4,4,20-Trimethyl-21-(4-methylphenoxy)pregna-8,14-di
en-3-ol. M.p. 118-124.degree. C.; [.alpha.].sub.D.sup.20 =-49.6.degree.
(c=0.5, dioxane). The product contained 15% of
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(4-methylphenoxy)pregna-6,8(14)
-dien-3-ol.
EXAMPLE 6
(3.beta.,5.alpha.,20S)-4,4,20-Trimethyl-21-(phenylthio)pregna-8,14-dien-3-o
l
i)--Following procedures analogous to those described under i-iv of Example
2, (3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-8,14-diene-3,21-diol
3-benzoate (Example 4, step viii) was converted to
(3.beta.,5.alpha.,20S)-3-[(1-ethoxyethyl)oxy]-4,4,20-trimethyl-pregna-8,14
-dien-21-ol.
ii)--p-Toluenesulfonic anhydride (4.56 g) was added to a solution of
(3.beta.,5.alpha.,20S)-3-[(1-ethoxyethyl)oxy]-4,4,20-trimethylpregna-8,14-
dien-21-ol (3.0 g) in dry pyridine (28 ml). The reaction mixture was
stirred at room temperature for 2 h and then poured into water. The
product was extracted into ethyl acetate; the combined organic phases were
washed with brine, dried over sodium sulfate and concentrated, to give
(3.beta.,5.alpha.,20S)-3-[(1-ethoxyethyl)oxy]-4,4,20-trimethyl-21-[[(4-met
hylphenyl)sulfonyl]oxy]pregna-8,14-diene (4.32 g), which was used in the
following step without further purification.
iii)--Sodium hydride (0.302 g) was added to a solution of the tosylate
described in the previous step (4.03 g) in dry dimethylformamide (40 ml).
Thiophenol (0.81 ml) was added and the reaction mixture was stirred for 30
min. at room temperature. The mixture was poured into water and the
product was extracted into ethyl acetate. The combined organic phases were
washed with brine, dried over sodium sulfate and concentrated. Column
chromatography afforded
(3.beta.,5.alpha.,20S)-3-[(1-ethoxyethyl)oxy]-4,4,20-trimethyl-21-(phenylt
hio)pregna-8,14-diene (3.0 g).
iv)--Following a procedure analogous to that described under iv of Example
1, the thioether described in the previous step (1.0 g) was converted into
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylthio)pregna-8,14-dien-3-
ol (0.24 g). M.p. 101-107.degree. C. The product contained 25% of
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylthio)pregna-6,8(14)-dien
-3-ol.
EXAMPLE 7
[3.beta.,5.alpha.,20S,(21R)]-4,4,20-Trimethyl-21-(phenylsulfinyl)pregna-8,1
4-dien-3-ol and
[3.beta.,5.alpha.,20S,(21S)]-4,4,20-trimethyl-21-(phenylsulfinyl)pregna-8,
14-dien-3-ol
i)--Following a procedure analogous to that described under ii of Example
6, (3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-8,14-diene-3,21-diol
3-benzoate (Example 4, step viii, 2.0 g) was converted to
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-[[(4-methylphenyl)sulfonyl]oxy]
pregna-8,14-dien-3-ol benzoate (2.95 g).
ii)--Following a procedure analogous to that described under iii of Example
6, the tosylate obtained in the previous step (2.95 g) was converted to
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylthio)pregna-8,14-dien-3-
ol benzoate (1.72 g).
iii)--Hydrogen peroxide (30%, 0.6 ml) was added to a solution of the
thioether described in the previous step (1.67 g) in dichloromethane (15
ml), followed by trifluoroacetone (0.054 ml). The mixture was stirred at
room temperature for 6 h and then poured into a saturated aqueous solution
of sodium thiosulfate. The product was extracted into dichloromethane; the
combined organic phases were washed with water, dried over sodium sulfate
and concentrated, to give a mixture of
[3.beta.,5.alpha.,20S,(21R)]-4,4,20-trimethyl-21-(phenylsulfinyl)pregna-8,
14-dien-3-ol benzoate and
[3.beta.,5.alpha.,20S,(21S)]-4,4,20-trimethyl-21-(phenylsulfinyl)pregna-8,
14-dien-3-ol benzoate (1.73 g), which was used in the following step
without further purification.
iv)--Potassium hydroxide (0.464 g) was added to a solution of the mixture
of sulfoxides obtained in the previous step (1.73 g) in a mixture of
tetrahydrofuran (10.7 ml) and methanol (1.25 ml). The reaction mixture was
stirred at room temperature for 2 h and then poured into water. The
product was extracted into ethyl acetate; the combined organic phases were
washed with water and brine, dried over sodium sulfate and concentrated.
Column chromatography of the crude product and crystallization afforded
[3.beta.,5.alpha.,20S,(21R)]-4,4,20-trimethyl-21-(phenylsulfinyl)pregna-8,
14-dien-3-ol (0.16 g), m.p. 185-191.degree. C., containing 20% of
[3.beta.,5.alpha.,20S,(21R)]-4,4,20-trimethyl-21-(phenylsulfinyl)pregna-6,
8(14)-dien-3-ol, and [3.beta.,5.alpha.,
20S,(21S)]-4,4,20-trimethyl-21-(phenylsulfinyl)pregna-8,14-dien-3-ol
(0.306 g), m.p. 187-201.degree. C.
EXAMPLE 8
(3.beta.,5.alpha.,20S)-4,4,20-Trimethyl-21-(phenylsulfonyl)pregna-8,14-dien
-3-ol
i)--Iodine (1.87 g) was added to a mixture of triphenylphosphine (2.04 g),
imidazole (0.53 g) and dry dichloromethane (48 ml). After 10 min. stirring
(3.beta.,5.alpha.,20S)-4,4,20-trimethylpregna-8,14-diene-3,21-diol
3-benzoate (Example 4, step viii, 2.0 g) in dry dichloromethane (20 ml)
was added and stirring was continued for 1 h. The reaction mixture was
poured into a saturated aqueous solution of sodium thiosulfate and the
product was extracted into dichloromethane. The combined organic phases
were washed with brine, dried over sodium sulfate and concentrated. Column
chromatography afforded
(3.beta.,5.alpha.,20S)-21-iodo-4,4,20-trimethylpregna-8,14-dien-3-ol
benzoate (2.29 g).
ii)--Sodium benzenesulfinate (0.74 g) was added to a solution of the iodide
obtained in the previous step (1.72 g) in dry dimethylformamide (7.5 ml).
The reaction mixture was stirred at 74.degree. C. for 3 h, cooled, and
poured into water. The product was extracted into ethyl acetate; the
combined organic phases were washed with brine, dried over sodium sulfate
and concentrated. Column chromatography gave
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylsulfonyl)pregna-8,14-die
n-3-ol benzoate (0.599 g).
iii)--Following a procedure analogous to that described under step iv of
Example 7, the sulfone obtained in the previous step (0.809 g) was
converted to
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylsulfonyl)pregna-8,14-die
n-3-ol (0.399 g). M.p. 175-188.degree. C. The product contained 25% of
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenylsulfonyl)pregna-6,8(14)-
dien-3-ol.
EXAMPLE 9
The Oocyte Assay
General
Oocytes arrested in meiosis contain diffused chromosomes which are
surrounded by an intact nuclear envelope known as the germinal vesicle
(GV). Upon reinitiation of meiosis by the midcycle gonadotropin surge, the
chromosomes recondense and the GV breaks down (GVBD). In vivo, the oocyte
is exposed to hypoxanthine (HX), which maintains the oocyte arrested in
the meiotic prophase. This meiotic arrest can be mimicked in vitro by
addition of hypoxanthine to the culture medium. The agonistic activity of
the compounds of the invention is measured as the ability to overcome the
hypoxanthine maintained meiotic arrest in denuded oocytes (DO) or cumulus
enclosed oocytes (CEO), i.e. as the ability to induce meiotic resumption
in vitro.
Therefore, natural resumption of meiosis can be mimicked in vitro by
addition of FF-Mas or other agonists to the culture medium containing
hypoxanthine. The antagonistic activity of the compounds is measured as
the ability to inhibit the FF-Mas or 22S-hydroxy-FF-Mas induced oocyte
maturation in denuded oocytes or cumulus enclosed oocytes, respectively,
in vitro.
Isolation of Cumulus Enclosed Oocytes
Ovaries are obtained from immature female mice (B6D2-F1, strain
C57BL.times.DBA). At the age of 19, 20 or 21 days the mice are injected
subcutaneously with a single dose of 20 IU follicle stimulating hormone
(Humegon, Organon, The Netherlands) in saline.
Forty-eight hours after follicle stimulating hormone injection mice are
killed by cervical dislocation. The ovaries are removed, freed of
extraneous tissue and placed in a multidish containing 0.5 ml preparation
medium at 37.degree. C. L-15 Leibovitz medium (Gibco, pH 7.3.+-.0.1)
supplemented with bovine serum albumin (3 mg.ml.sup.-1), L-glutamine (0.23
mM), sodium pyruvate (2 mM) and hypoxanthine (4 mM) is used as preparation
medium. The antral follicles of the ovaries are punctured under a
dissecting microscope using two 27-gauge needles attached to two 1 ml
syringes. Cumulus enclosed oocytes (CEO) of uniform size are selected with
a mouth-controlled pipette and rinsed in 0,5 ml fresh preparation medium.
About 20 CEO are obtained from one ovary.
Isolation of Denuded Oocytes
Oocytes freed from cumulus cells, i.e. denuded oocytes (DO), are obtained
by gently flushing CEO through a fine-bore mouth-controlled pipette. DO
were collected in fresh MEM alpha medium, containing bovine serum albumin
(3 mg.ml.sup.-1), L-glutamine (0.23 mM), sodium pyruvate (2 mM) and
hypoxanthine (4 mM), and washed twice before transfer to the test medium.
Experimental Design
The oocyte assay is performed in 3 blocks, each block represents the
ovaries of one mouse (randomized block design). At t=0 DO or CEO of the
first ovary of the first mouse, are spread over well 1 and 3 and oocytes
of the second ovary over well 2 and 4 of a 4-well multidish containing 0.5
ml of culture medium to which a
17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative of the
invention is added in case of agonistic testing. In case of antagonistic
testing, a 17.beta.-aryl(arylmethyl)oxy(thio)alkyl-androstane derivative
of the invention is added together with FF-Mas (DO assay) or
22S-hydroxy-FF-Mas (CEO assay) [first block]. In case of agonistic
testing, culture medium was used as control; in case of antagonistic
testing, culture medium containing FF-Mas (DO assay) or 22S-hydroxy-FF-Mas
(CEO assay) was used as control. The same procedure is performed for the
second and third mouse [block 2 and 3]. The culture medium used is MEM
alpha medium (Gibco, pH 7.3.+-.0.1) saturated with CO.sub.2 and
supplemented with bovine serum albumin (3 mg.ml.sup.-1), L-glutamine (0.23
mM), sodium pyruvate (2 mM) and hypoxanthine (4 mM). In total, each
control or test compound is tested on 30 oocytes (10 oocytes per block).
At t=0 the number of oocytes with intact germinal vesicles (GV) or
germinal vesicle break-down (GVBD) is counted under an inverted microscope
with differential interference contrast equipment. Only oocytes with an
intact GV are used in the experiment. Oocytes are cultured 22 hours at
37.degree. C. in 100% humidified atmosphere with 5% CO.sub.2 in air. At
the end of the culture period the number of oocytes with GV or GVBD per
group is counted. For statistical analysis the percentage germinal vesicle
breakdown is calculated for each group in one block. These percentages are
subjected to arcsin transformation, and differences between control and
test compounds are analyzed by an ANOVA test for a randomized block
design. Results are presented in Table I (agonists) and II (antagonists).
TABLE I
Percentage germinal vesicle breakdown (GVBD) in oocytes following
culturing in the presence of test compounds in agonistic testing.
% GVBD
DO assay CEO assay
Compound* exp.(control) exp.(control)
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-phenoxypreg- 100(0) 96(0)
na-8,14-dien-3-ol (Example 1)
(3.beta.,5.alpha.,20R)-4,4-dimethyl-20-(phenylmeth- 100(11) 59(0)
oxy)-pregna-8,14-dien-3-ol (Example 2)
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-phenoxy- 72(3) 1(0)
pregna-8,14-dien-3-ol (Example 4)
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(2-methyl- 100(6) 7(0)
phenoxy)pregna-8,14-dien-3-ol
(Example 5A)
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(4-methyl- 87(3) 16(4)
phenoxy)pregna-8,14-dien-3-ol
(Example 5B)
(3.beta.,5.alpha.,20R)-4,4-dimethylcholesta-8,14,24- 84(7) 0(0)
trien-3-ol (FF-Mas)
(3.beta.,5.alpha.,20S,22S)-4,4-dimethylcholesta-8,14, 97(2) 73(2)
24-triene-3,22-diol (22S-hydroxy-FF-Mas)**
*Each compound was tested at a concentration of 10 .mu.M.
**Tested at a concentration of 5 .mu.M.
TABLE II
Percentage germinal vesicle breakdown (GVBD) in oocytes following
culturing in the presence of test compounds and FF-Mas (DO assay)
or 22S-hydroxy-FF-Mas (CEO assay) in antagonistic testing.
% GVBD
DO assay* CEO assay**
Compound exp.(control) exp.(control)
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenyl- 64(94)
thio)pregna-8,14-dien-3-ol (Example 6)
(3.beta.,5.alpha.,20S)-4,4,20-trimethyl-21-(phenyl- 32(84) 14(89)
sulfonyl)pregna-8,14-dien-3-ol
(Example 8)
*Concentration of
(3.beta.,5.alpha.,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol (FF-Mas): 5
.mu.M; concentration of compound tested: 5 .mu.M.
**Concentration of
(3.beta.,5.alpha.,20S,22S)-4,4-dimethylcholesta-8,14,24-triene-3,22-diol
(22S-hydroxy-FF-Mas): 5 .mu.M; concentration of compound tested: 2.5
.mu.M.
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